Rhodopsin is the photoreceptor responsible for dim light vision, and represents the paradigm for the large superfamily of G protein-coupled receptors (GPCRs). The importance of rhodopsin in vision and in medicine is demonstrated by the fact that its mutants are linked to various retinal dystrophies, and mutations causing constitutive activity of rhodopsins are linked to congenital stationary night blindness. Rhodopsin has the canonical seven transmembrane helices, which delineate the pocket for the 11-cis retinal chromophore that is bound to Lys296 via a protonated Schiff base. The X-ray structure of ground-state bovine rhodopsin has been elucidated to 2.8A resolution. Determining this high-resolution X-ray structure was a major step toward developing an understanding of rhodopsin's function; however, elucidating its mechanism of activation requires knowing the high-resolution structures of intermediates along the reaction pathway, as well as their modes of interaction with transducin. Specifically, we will ask how does retinal photoisomerization trigger protein structural changes that lead to activation of transducin, thereby initiating the sensation of vision. Our major hypothesis is that light triggers the rapid isomerization of the retinal, a change which the protein both accommodates and subsequently amplifies into the larger structural changes required for signaling. We are in a unique position to address this hypothesis, as we have recently achieved a higher resolution structure of rhodopsin and have for the first time identified internal water molecules unambiguously. Furthermore, we have trapped photo-intermediates of rhodopsins in the crystals and shown that they diffract. The aims of this project are to map, at unprecedented spatial and temporal resolution, the structural transformations that occur upon activation of rhodospin by solving the X-ray structures of the photo-intermediates bathorhodopsin, lumirhodopsin and metarhodopsin (The latter is the signaling state of rhodopsin). Most importantly, we will elucidate the crystal structure of the metarhodopsin-transducin complex in order to identify the interactions at the interface of this signaling unit. Accomplishing these Specific Aims will define the physical and chemical basis for the activation of rhodopsin. Furthermore, structural studies at high resolution will open the way for understanding the molecular basis for the unique photobleaching properties of rod and cone visual pigments.